Sm0020

The experimental animals were deeply anesthetized and transcardially perfused with ice-cold 0.9% saline solution. Following perfusion, half of the brain extracted from the skull was stored at −80 °C and prepared for protein and mitochondrial analysis. The other half of the brain was postfixed in freshly prepared 4% paraformaldehyde (PFA) overnight at 4 °C and then dehydrated in a gradient of 20% and 30% sucrose and 0.1 M phosphate-buffered saline (PBS, pH = 7.4) until it sank and embedded in tissue embedding medium (4583, OCT Compound; Sakura Finetek Tissue-Tek, Tokyo, Japan). Coronal sections (10 μm thick) encompassing the entire cortex and hippocampus were cut with a freezing microtome (CM1860; Leica, Wetzlar, Germany), collected sequentially in 6-well plates filled with 0.01 M PBS and mounted onto slides for immunofluorescence analysis. Uteri were dissected and weighed. The manufacturer's instructions for the mitochondrial extraction kit (SM0020, Beijing Solarbio Science & Technology Co., Ltd.) were followed closely. We obtained high purity mitochondria by the differential centrifugation method. It is worth noting that all of the abovementioned procedures should be performed at a low temperature.

The ER, mitochondria (Mito.), and Golgi apparatus were isolated with an ER isolation kit (Solarbio, EX2690), mitochondria isolation kit (Solarbio, SM0020), and Golgi apparatus isolation kit (Solarbio, EX1360-100T) according to the manufacturer’s protocol. Mitochondria-associated membrane was isolated following the protocol as reported previously (79 ).

The prefrontal cortices were homogenized in a 10-fold volume lysis buffer (SM0020, Solarbio, Beijing, China). The homogenate was then centrifuged at 1000× g for 5 min, and the supernatants were centrifuged at 1000× g for another 5 min. Next, the supernatants were centrifuged at 12,000× g for 10 min, and then collected and used as cytoplasmic extracts. The precipitate was then resuspended in wash buffer and centrifuged at 1000× g for 5 min. The supernatant was centrifuged again at 12,000× g for 10 min. Finally, the precipitate was resuspended in store buffer and considered a highly pure mitochondrial extract, which was used to detect the level of MMP and the activity of mitochondrial respiratory chain complexes.

The mitochondrial and cytoplasmic proteins of the prefrontal cortex were extracted using an appropriate kit (SM0020, Solarbio) and quantified using a BCA kit (CW0014S, CWBIO, Beijing, China). Protein samples were mixed with loading buffer and boiled at 99 °C for 5 min. Protein samples (10 μg) were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Billerica, MA, USA) for blocking in 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were incubated with polyclonal rabbit primary antibodies in blocking solution at 4 °C overnight, including anti-GAPDH (1:60.000, KC-5G5, Kangchen, Shanghai, China) and anti-COXIV (1:1000, 4844, Cell Signaling Technology, Danvers, MA, USA) antibodies. The membranes were then washed three times with PBST and incubated with goat anti-rabbit secondary antibody (1:120,000, KC-RB-025, Kangchen) for 30 min at room temperature. Immunoblotting bands were visualized by incubation with ECL reagent (WBLUF0100, Merck Millipore, Darmstadt, Germany) and exposed to an X-ray film (6535876, Kodak, Rochester, NY, USA).